Although the extracellular matrix (ECM) has been viewed as a static support structure, our recent studies using time lapse computational imaging have suggested that bone ECM proteins are highly dynamic and are constantly subjected to stretching, contracting and reorganization in response to cell motion. Fibronectin is an ECM glycoprotein that acts as an orchestrator for assembly of multiple bone matrix proteins, including type I collagen. Our preliminary data suggest that the assembly of fibronectin and type I collagen are spatially and temporally integrated. These studies have also revealed novel mechanisms for ECM assembly and reorganization, including shunting of fibrillar material from one location to another by motile cells and exchange of fibrillar material between fibrils. We have also shown that fibronectin plays a key role in mineralization in vitro. Although there has been an increased understanding of the molecular mechanisms for bone mineralization and its dependence on collagen, little is known about the dynamics of the mineralization process and how ECM protein dynamics are integrated with the process of mineralization. The underlying hypothesis for the proposed studies is that "Fibronectin and type I collagen assembly is a dynamic process that must be spatially and temporally integrated to form an appropriate substrate for biomineralization". To address this hypothesis I will use state-of-the-art live cell imaging techniques in which fibronectin and type I collagen are simultaneously imaged in mineralizing osteoblast cultures using fluorescent probes. I will determine how ECM assembly and reorganization are dynamically integrated with mineralization and determine the role of cell motility in these processes. This will be combined with computational approaches to measure ECM fibril kinetics. These studies will provide novel insights into the mechanisms of bone matrix assembly and reorganization and will enhance our understanding of the processes by which the ECM regulates biomineralization in bone development and disease. [unreadable] [unreadable] [unreadable]